The primary structure of rubrerythrin, a protein with inorganic pyrophosphatase activity from Desulfovibrio vulgaris. Comparison with hemerythrin and rubredoxin.

The complete polypeptide chain of rubrerythrin from the sulfate reducing bacterium Desulfovibrio vulgaris, strain Hildenborough NCIB 8303, was found by protein chemical techniques to consist of 191 residues and to have the amino acid sequence [sequence: see text] The C-terminal part of the protein (position 153----191) shows the typical sequence features of rubredoxin, a protein with a nonheme iron center also present in the same and other Desulfovibrio species. Based on the known three-dimensional structure of D. desulfuricans rubredoxin, we propose that the C-terminal part of rubrerythrin is folded in a similar way and suggest that the deletion of the extra 10 residues is compatible with the same basic rubredoxin-fold. After characterization of the C-terminal region, and in contrast to what could be expected from previously published spectroscopic analyses, the N-terminal region 1-152 of rubrerythrin appears to have no sequence similarity with the eukaryotic protein hemerythrin which is known to contain a binuclear iron center bound by 5 histidine ligands. However, the N-terminal region of rubrerythrin does contain 5 histidine residues but they are differently spaced along the peptide chain. We suggest that at least one of the 3 histidine residues located in the rubredoxin-like center of rubrerythrin may be liganded to one iron atom of the hemerythrin-like center. This paper is the first sequence report of a protein with pyrophosphatase activity although the physiological substrate for the rubrerythrin may be not inorganic pyrophosphate.     

Structure,function and evolution of the hemerythrin‐like domain superfamily

Hemerythrin‐like proteins have generally been studied for their ability to reversibly bind oxygen through their binuclear nonheme iron centers. However, in recent years, it has become increasingly evident that some members of the hemerythrin‐like superfamily also participate in many other biological processes. For instance, the binuclear nonheme iron site of YtfE, a hemerythrin‐like protein involved in the repair of iron centers in Escherichia coli, catalyzes the reduction of nitric oxide to nitrous oxide, and the human F‐box/LRR‐repeat protein 5, which contains a hemerythrin‐like domain, is involved in intracellular iron homeostasis. Furthermore, structural data on hemerythrin‐like domains from two proteins of unknown function, PF0695 from Pyrococcus furiosus and NMB1532 from Neisseria meningitidis, show that the cation‐binding sites, typical of hemerythrin, can be absent or be occupied by metal ions other than iron. To systematically investigate this functional and structural diversity of the hemerythrin‐like superfamily, we have collected hemerythrin‐like sequences from a database comprising fully sequenced proteomes and generated a cluster map based on their all‐against‐all pairwise sequence similarity. Our results show that the hemerythrin‐like superfamily comprises a large number of protein families which can be classified into three broad groups on the basis of their cation‐coordinating residues: (a) signal‐transduction and oxygen‐carrier hemerythrins (H‐HxxxE‐HxxxH‐HxxxxD); (b) hemerythrin‐like (H‐HxxxE‐H‐HxxxE); and, (c) metazoan F‐box proteins (H‐HExxE‐H‐HxxxE). Interestingly, all but two hemerythrin‐like families exhibit internal sequence and structural symmetry, suggesting that a duplication event may have led to the origin of the hemerythrin domain.     

Anion-independent iron coordination by the Campylobacter jejuni ferric binding protein

Campylobacter jejuni, the leading cause of human gastroenteritis, expresses a ferric binding protein (cFbpA) that in many pathogenic bacteria functions to acquire iron as part of their virulence repertoire. Recombinant cFbpA is isolated with ferric iron bound from Escherichia coli. The crystal structure of cFbpA reveals unprecedented iron coordination by only five protein ligands. The histidine and one tyrosine are derived from the N-terminal domain, whereas the three remaining tyrosine ligands are from the C-terminal domain. Surprisingly, a synergistic anion present in all other characterized ferric transport proteins is not observed in the cFbpA iron-binding site, suggesting a novel role for this protein in iron uptake. Furthermore, cFbpA is shown to bind iron with high affinity similar to Neisserial FbpA and exhibits an unusual preference for ferrous iron (oxidized subsequently to the ferric form) or ferric iron chelated by oxalate. Sequence and structure analyses reveal that cFbpA is a member of a new class of ferric binding proteins that includes homologs from invasive and intracellular bacteria as well as cyanobacteria. Overall, six classes are defined based on clustering within the tree and by their putative iron coordination. The absence of a synergistic anion in the iron coordination sphere of cFbpA also suggests an alternative model of evolution for FbpA homologs involving an early iron-binding ancestor instead of a requirement for a preexisting anion-binding ancestor.     

Schistosoma mansoni: SmLIMPETin, a member of a novel family of invertebrate-only regulatory proteins

Eukaryotic LIM domain proteins contain zinc finger forming motifs rich in cysteine and histidine that enable them to interact with other proteins. A cDNA clone isolated from an adult schistosome cDNA library revealed a sequence that coded for a novel class of proteins bearing 6 LIM domains and an N-terminal PET domain, SmLIMPETin. Phylogeny reconstruction of SmLIMPETin and comparison of its sequence to invertebrate homologues and to the vertebrate four-and-a-half LIM domains protein family (FHLs), uncovered a novel LIM domain protein family, the invertebrate LIM and PET domain protein family (LIMPETin). Northern blots, RT-PCR and Western blot showed that SmLIMPETin gene was less expressed in sexually mature adult females compared to sexually immature adult females and sexually mature and immature adult males, and not expressed in schistosomula.     

Crystal structure of hexameric haemocyanin from Panulirus interruptus refined at 3.2 A resolution

The use of non-crystallographic symmetry restraints in the refinement of the haemocyanin hexamer from Panulirus interruptus at 3.2 A resolution has resulted in a final model with a very reasonable geometry and a crystallographic R-factor of 20.1%, using 59,193 observed structure factor amplitudes between 8.0 and 3.2 A. The mean co-ordinate error is approximately 0.35 A. The six subunits appear to be related by symmetry operations that differ slightly from 32 point group symmetry. The six subunits have essentially maintained the same structure. The hexamer, with point group 32, is best described as a trimer of "tight dimers". The contacts between the subunits in such a dimer are more numerous, and better conserved during evolution than contacts in a trimer. The interface of a tight dimer is separated by an internal cavity into two "contact areas". The contact area nearest to the centre of the hexamer is most extensive and consists mainly of residues that are quite conserved among arthropodan haemocyanins. All these residues are provided by the second domain of each subunit. Hence, this second domain may play a crucial role in the allosteric functioning of this oxygen transport protein. The dinuclear copper oxygen-binding site resides in the centre of domain 2. This oxygen-binding centre is not fully accessible from the solvent. Three large cavities occur, however, within each subunit at the interfaces of the three domains. All three cavities contain ordered water molecules, and two of them are accessible from the surrounding solvent. These cavities may play a role in facilitating fast movement of dioxygen towards the binding site, which is situated in a highly conserved, rather hydrophobic core. A detailed definition of the geometry of the copper site is, of course, not possible at the limited resolution of 3.2 A. Nevertheless, it is possible to conclude that each copper is co-ordinated by two, more or less tightly bound, histidine ligands and one more distant histidine residue. The six histidine residues utilize their N epsilon atoms for copper co-ordination, while their N delta atoms are engaged in hydrogen bonds with conserved residues or water molecules. The two distant histidine ligands are located in apical positions and are on opposite sides with respect to the plane approximately defined by the four more tightly bound histidine ligands and the two copper ions. The copper-to-copper distance is 3.5 to 3.6 A in four of the subunits, but this distance deviates considerably in two others.(ABSTRACT TRUNCATED AT 400 WORDS)     

Terminal proteins of Streptomyces chromosome can target DNA into eukaryotic nuclei

Streptomyces species are highly abundant soil bacteria that possess linear chromosomes (and linear plasmids). The 5' ends of these molecules are covalently bound by terminal proteins (TPs), that are important for integrity and replication of the telomeres. There are at least two types of TPs, both of which contain a DNA-binding domain and a classical eukaryotic nuclear localization signal (NLS). Here we show that the NLS motifs on these TPs are highly efficient in targeting the proteins along with covalently bound plasmid DNA into the nuclei of human cells. The TP-mediated nuclear targeting resembles the inter-kingdom gene transfer mediated by Ti plasmids of Agrobacterium tumefaciens, in which a piece of the Ti plasmid DNA is targeted to the plant nuclei by a covalently bound NLS-containing protein. The discovery of the nuclear localization functions of the Streptomyces TPs not only suggests possible inter-kingdom gene exchanges between Streptomyces and eukaryotes in soil but also provides a novel strategy for gene delivery in humans and other eukaryotes.     

Isolation, purification and characterization of hemerythrin from Methylococcus capsulatus (Bath)

Earlier work from our laboratory has indicated that a hemerythrin-like protein was over-produced together with the particulate methane monooxygenase (pMMO) when Methylococcus capsulatus (Bath) was grown under high copper concentrations. A homologue of hemerythrin had not previously been found in any prokaryote. To confirm its identity as a hemerythrin, we have isolated and purified this protein by ion-exchange, gel-filtration and hydrophobic interaction chromatography, and characterized it by mass spectrometry, UV-visible, CD, EPR and resonance Raman spectroscopy. On the basis of biophysical and multiple sequence alignment analysis, the protein isolated from M. capsulatus (Bath) is in accord with hemerythrins previously reported from higher organisms. Determination of the Fe content in conjunction with molecular-weight estimation and mass analysis indicates that the native hemerythrin in M. capsulatus (Bath) is a monomer with molecular mass 14.8 kDa, in contrast to hemerythrins from other eukaryotic organisms, where they typically exist as a tetramer or higher oligomers.     

Putative osmosensor – OsHK3b – a histidine kinase protein from rice shows high structural conservation with its ortholog AtHK1 from Arabidopsis

Prokaryotes and eukaryotes respond to various environmental stimuli using the two-component system (TCS). Essentially, it consists of membrane-bound histidine kinase (HK) which senses the stimuli and further transfers the signal to the response regulator, which in turn, regulates expression of various target genes. Recently, sequence-based genome wide analysis has been carried out in Arabidopsis and rice to identify all the putative members of TCS family. One of the members of this family i.e. AtHK1, (a putative osmosensor, hybrid-type sensory histidine kinase) is known to interact with AtHPt1 (phosphotransfer proteins) in Arabidopsis. Based on predicted rice interactome network (PRIN), the ortholog of AtHK1 in rice, OsHK3b, was found to be interacting with OsHPt2. The analysis of amino acid sequence of AtHK1 showed the presence of transmitter domain (TD) and receiver domain (RD), while OsHK3b showed presence of three conserved domains namely CHASE (signaling domain), TD, and RD. In order to elaborate on structural details of functional domains of hybrid-type HK and phosphotransfer proteins in both these genera, we have modeled them using homology modeling approach. The structural motifs present in various functional domains of the orthologous proteins were found to be highly conserved. Binding analysis of the RD domain of these sensory proteins in Arabidopsis and rice revealed the role of various residues such as histidine in HPt protein which are essential for their interaction.     

Iron economy in Naegleria gruberi reflects its metabolic flexibility

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.     

Histidine protein kinases: key signal transducers outside the animal kingdom

Histidine protein kinases (HPKs) are a large family of signal-transduction enzymes that autophosphorylate on a conserved histidine residue. HPKs form two-component signaling systems together with their downstream target proteins, the response regulators, which have a conserved aspartate in a so-called 'receiver domain' that is phosphorylated by the HPK. Two-component signal transduction is prevalent in bacteria and is also widely used by eukaryotes outside the animal kingdom. The typical HPK is a transmembrane receptor with an amino-terminal extracellular sensing domain and a carboxy-terminal cytosolic signaling domain; most, if not all, HPKs function as dimers. They show little similarity to protein kinases that phosphorylate serine, threonine or tyrosine residues, but may share a distant evolutionary relationship with these enzymes. In excess of a thousand known genes encode HPKs, which are important for multiple functions in bacteria, including chemotaxis and quorum sensing, and in eukaryotes, including hormone-dependent developmental processes. The proteins divide into at least 11 subfamilies, only one of which is present in eukaryotes, suggesting that lateral gene transfer gave rise to two-component signaling in these organisms.     

CASP,the alternatively spliced product of the gene encoding the CCAAT-displacement protein transcription factor,is a Golgi membrane protein related to giantin

Large coiled-coil proteins are being found in increasing numbers on the membranes of the Golgi apparatus and have been proposed to function in tethering of transport vesicles and in the organization of the Golgi stack. Members of one class of Golgi coiled-coil protein, comprising giantin and golgin-84, are anchored to the bilayer by a single C-terminal transmembrane domain (TMD). In this article, we report the characterization of another mammalian coiled-coil protein, CASP, that was originally identified as an alternatively spliced product of the CUTL1 gene that encodes CCAAT-displacement protein (CDP), the human homologue of the Drosophila homeodomain protein Cut. We find that the Caenorhabditis elegans homologues of CDP and CASP are also generated from a single gene. CASP lacks the DNA binding motifs of CDP and was previously reported to be a nuclear protein. Herein, we show that it is in fact a Golgi protein with a C-terminal TMD and shares with giantin and golgin-84 a conserved histidine in its TMD. However, unlike these proteins, CASP has a homologue in Saccharomyces cerevisiae, which we call COY1. Deletion of COY1 does not affect viability, but strikingly restores normal growth to cells lacking the Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor Gos1p. The conserved histidine is necessary for Coy1p's activity in cells lacking Gos1p, suggesting that the TMD of these transmembrane Golgi coiled-coil proteins is directly involved in their function.     

Functionally specified protein signatures distinctive for each of the different blue copper proteins

Background  

Proteins having similar functions from different sources can be identified by the occurrence in their sequences, a conserved cluster of amino acids referred to as pattern, motif, signature or fingerprint. The wide usage of protein sequence analysis in par with the growth of databases signifies the importance of using patterns or signatures to retrieve out related sequences. Blue copper proteins are found in the electron transport chain of prokaryotes and eukaryotes. The signatures already existing in the databases like the type 1 copper blue, multiple copper oxidase, cyt b/b6, photosystem 1 psaA&B, psaG&K, and reiske iron sulphur protein are not specified signatures for blue copper proteins as the name itself suggests. Most profile and motif databases strive to classify protein sequences into a broad spectrum of protein families. This work describes the signatures designed based on the copper metal binding motifs in blue copper proteins. The common feature in all blue copper proteins is a trigonal planar arrangement of two nitrogen ligands [each from histidine] and one sulphur containing thiolate ligand [from cysteine], with strong interactions between the copper center and these ligands.     

真核生物FANCJ-like蛋白的结构与进化

FANCJ-like蛋白是一类ATP依赖的5′-3′DNA解旋酶,参与DNA损伤修复、同源重组及G4-DNA拆解,在基因组稳定性维持过程中发挥重要功能。文章系统分析了47种真核生物的FANCJ-like蛋白,对其序列结构特征及起源进化进行了深入探讨。真核生物FANCJ-like蛋白包含4类成员——XPD、CHL1、RTEL1和FANCJ,但在真菌的一些世系及昆虫中存在严重缺失现象,如接合菌门(Zygomycota)缺失了RTEL1,担子菌门(Basidiomycota)和子囊菌门(Ascomycota)缺失了RTEL1和FANCJ,双翅目昆虫缺失了FANCJ。FANCJ-like蛋白不仅包含经典解旋酶共有HD1和HD2结构域,而且在HD1结构域中插入了自身特有的Fe-S、Arch和Extra-D结构域。Fe-S和Arch结构域在4类成员中较保守,Extra-D结构域在XPD中不存在,在其他3类成员中也各不相同。在FANCJ-like蛋白的Fe-S、Arch和Extra-D结构域中分别发现了7个、10个和2个特有模体;除了已报道的保守模体外,HD1和HD2中分别发现了5个和12个特有模体。从这些特有模体的组成和排布来看,RTEL1和FANCJ最为相近,它们在HD2区包含两个独有模体Vb2和Vc,可能与其G4-DNA解旋活性相关。进化方面的证据表明,FANCJ-like蛋白起源于一种HD1区插入了Fe-S和Arch结构域的DNA解旋酶,在多细胞真核生物出现之前,该蛋白通过3次复制事件和随后的特异化过程,依次形成了目前真核生物所包含的4类FANCJ-like蛋白。     

Oxypred: prediction and classification of oxygen-binding proteins

This study describes a method for predicting and classifying oxygen-binding proteins. Firstly, support vector machine (SVM) modules were developed using amino acid composition and dipeptide composition for predicting oxygen-binding proteins, and achieved maximum accuracy of 85.5% and 87.8%, respectively. Secondly, an SVM module was developed based on amino acid composition, classifying the predicted oxygen-binding proteins into six classes with accuracy of 95.8%, 97.5%, 97.5%, 96.9%, 99.4%, and 96.0% for erythrocruorin, hemerythrin, hemocyanin, hemoglobin, leghemoglobin, and myoglobin proteins, respectively. Finally, an SVM module was developed using dipeptide composition for classifying the oxygen-binding proteins, and achieved maximum accuracy of 96.1%, 98.7%, 98.7%, 85.6%, 99.6%, and 93.3% for the above six classes, respectively. All modules were trained and tested by five-fold cross validation. Based on the above approach, a web server Oxypred was developed for predicting and classifying oxygen-binding proteins (available from http://www.imtech.res.in/raghava/oxypred/).     

The structure of the iron-binding protein, FutA1, from Synechocystis 6803

Cyanobacteria account for a significant percentage of aquatic primary productivity even in areas where the concentrations of essential micronutrients are extremely low. To better understand the mechanism of iron selectivity and transport, the structure of the solute binding domain of an ATP binding cassette iron transporter, FutA1, was determined in the presence and absence of iron. The iron ion is bound within the "C-clamp" structure via four tyrosine and one histidine residues. There are extensive interactions between these ligating residues and the rest of the protein such that the conformations of the side chains remain relatively unchanged as the iron is released by the opening of the metal binding cleft. This is in stark contrast to the zinc-binding protein, ZnuA, where the domains of the metal-binding protein remain relatively fixed, whereas the ligating residues rotate out of the binding pocket upon metal release. The rotation of the domains in FutA1 is facilitated by two flexible beta-strands running along the back of the protein that act like a hinge during domain motion. This motion may require relatively little energy since total contact area between the domains is the same whether the protein is in the open or closed conformation. Consistent with the pH dependence of iron binding, the main trigger for iron release is likely the histidine in the iron-binding site. Finally, neither FutA1 nor FutA2 binds iron as a siderophore complex or in the presence of anions, and both preferentially bind ferrous over ferric ions.     

Comparison between site-specific DNA binding proteins of male and female Schistosoma mansoni

Several amplicons with approximately 120 bp each, obtained from the upstream domain of Schistosoma mansoni female-specific gene F-10, were coupled to Dynabeads M-280 streptavidin. The beads were used as a matrix for affinity purification of nuclear proteins obtained from mixed populations of adult worms. A protein of approximately 12 kDa, bound to the DNA in a sequence-independent manner. In contrast, when the DNA matrix was narrowed down to smaller synthetic oligonucleotides, bearing sequences corresponding to the TATA box and the CAAT box, band-shift assays revealed that different nuclear proteins from either adult male or female worms formed complexes with the DNA adduct. In order to characterise the bound proteins, the same oligonucleotides were UV cross-linked to the male and female protein extracts. Whilst the band shift experiments showed that the proteins from each sex produced a distinct mobility pattern when the TATA box sequences were tested and a similar one when the CAAT box sequences were added to the proteins, UV cross-linking experiments revealed clear qualitative differences between both, male and female proteins and also between the proteins binding to the two motifs. These results are compatible with a model in which the differential expression of the F-10 gene might depend on individual sub-sets of proteins.     

Prediction of an HMG-box fold in the C-terminal domain of histone H1: insights into its role in DNA condensation

In eukaryotes, histone H1 promotes the organization of polynucleosome filaments into chromatin fibers, thus contributing to the formation of an important structural framework responsible for various DNA transaction processes. The H1 protein consists of a short N-terminal "nose," a central globular domain, and a highly basic C-terminal domain. Structure prediction of the C-terminal domain using fold recognition methods reveals the presence of an HMG-box-like fold. We recently showed by extensive site-directed and deletion mutagenesis studies that a 34 amino acid segment encompassing the three S/TPKK motifs, within the C-terminal domain, is responsible for DNA condensing properties of H1. The position of these motifs in the predicted structure corresponds exactly to the DNA-binding segments of HMG-box-containing proteins such as Lef-1 and SRY. Previous analyses have suggested that histone H1 is likely to bend DNA bound to the C-terminal domain, directing the path of linker DNA in chromatin. Prediction of the structure of this domain provides a framework for understanding the higher order of chromatin organization.     

LRP130, a pentatricopeptide motif protein with a noncanonical RNA-binding domain,is bound in vivo to mitochondrial and nuclear RNAs

LRP130 (also known as LRPPRC) is an RNA-binding protein that is a constituent of postsplicing nuclear RNP complexes associated with mature mRNA. It belongs to a growing family of pentatricopeptide repeat (PPR) motif-containing proteins, several of which have been implicated in organellar RNA metabolism. We show here that only a fraction of LRP130 proteins are in nuclei and are directly bound in vivo to at least some of the same RNA molecules as the nucleocytoplasmic shuttle protein hnRNP A1. The majority of LRP130 proteins are located within mitochondria, where they are directly bound to polyadenylated RNAs in vivo. In vitro, LRP130 binds preferentially to polypyrimidines. This RNA-binding activity maps to a domain in its C-terminal region that does not contain any previously described RNA-binding motifs and that contains only 2 of the 11 predicted PPR motifs. Therefore, LRP130 is a novel type of RNA-binding protein that associates with both nuclear and mitochondrial mRNAs and as such is a potential candidate for coordinating nuclear and mitochondrial gene expression. These findings provide the first identification of a mammalian protein directly bound to mitochondrial RNA in vivo and provide a possible molecular explanation for the recently described association of mutations in LRP130 with cytochrome c oxidase deficiency in humans.